Necrotizing enterocolitis (NEC), an inflammatory bowel necrosis that primarily afflicts preterm infants after the initiation of enteral feeds, is likely the result of synergy of several risk factors including altered intestinal barrier function leading to an exaggerated inflammatory response to gut microbial flora as well as altered intestinal blood flow regulation leading to ischemia and tissue injury from oxidant-mediated damage. In response to a NIH program announcement (PA-06-316) for investigations into mechanisms of probiotic formulation for treatment of pediatric illnesses, we propose to test the hypothesis that conditioned media (CM) obtained by filtering the broth used to grow the probiotic bacteria Lactobacillus plantarum can decrease the incidence and severity of NEC by improving intestinal host defense mechanisms, including (A) enhancement of intestinal barrier function, (B) induction of cytoprotective heat shock proteins and protection against ischemia-mediated oxidant injury, and (C) modulation of intestinal inflammatory responses by inhibition of NF-kB activation/proteasome function. To control for specificity of effect, conditioned media from other gut bacteria which do not display the same type of bioactivity (e.g. E.coli) will be used as controls. As stated in the program announcement, the intent of this proposal is to utilize a complex formulation of natural bioactive components synthesized and secreted by the probiotics in a solution that is bacteria-free. We will test our hypothesis through the following specific aims: (1) Establish that probiotic CM (instead of live probiotic bacteria) decreases the incidence and severity of NEC by using a rat NEC model. (2) Investigate the mechanism of CM protection. Specifically, we will evaluate heat shock protein production (Hsp) by Western blot and ELISA, and also evaluate differences in oxidant-mediated injury by measuring oxidant markers such as xanthine oxidase, malondialdehyde, and nitric oxide production. Barrier function will be measured by in vivo permeability studies using FITC-dextran. Expression of specific tight junction proteins will be measured by confocal microscopy and western blot. L.plantarum CM-induced NF-kB/proteasome inhibition will be measured by a sequential evaluation of the NF-kB signaling pathway including NF-kB binding activity and nuclear localization, IkB degradation and ubiquitination, proteasome activity, and cytokine release. We propose that L. plantarum-CM, which is bacteria-free and thus safer to use, will have the capacity to alter the balance of intestinal cytoprotective and pro-inflammatory influences and thus protect against intestinal injury in NEC. Since there is no treatment for this devastating disease, limiting the initiation or propagation of intestinal injury and understanding the mechanisms involved could significantly improve outcome. PUBLIC HEALTH RELEVANCE: There is no specific treatment for necrotizing enterocolitis (NEC), and while clinical trials suggest that probiotics are protective, no mechanism has been delineated. Furthermore, bioactive factors in bacteria-free probiotic CM may provide a safer alternative to conventional probiotic use. Understanding how probiotic CM may limit the initiation or propagation of intestinal injury in this devastating disease could significantly improve outcome.